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Aims: Epidermal growth factor receptor (EGFR) antagonists are particularly active in non-small cell lung cancer (NSCLC) patients with tumours bearing mutations in the EFGR gene. EGFR mutation prevalence is very low in squamous histology. Response rates using these drugs in patients with KRAS mutations are low, so available KRAS mutation information may aid treatment selection in the second-line setting. Since 2009, patients presenting to this hospital with non-squamous histology have been routinely screened for mutations in both the EGFR and KRAS genes, with results used to inform treatment. We present an analysis of 215 consecutive patients for whom EGFR mutation analysis was informative. Methodology: EGFR and KRAS mutations were identified using a COLD-PCR technique confirmed with sequencing, which makes no prior assumption about location of specific mutations. Results were correlated with clinical and demographic data from hospital records, where available. Results: The prevalence of patients with EGFR mutations was 14% and for KRAS mutations it was 27%. Despite the conventional understanding that EGFR and KRAS mutations are mutually exclusive, we identified two dual mutations. Of 29 patients identified with mutated EGFR, there were 3/8/8/10 mutations in exons 18/19/20/21 respectively. Exon 20 mutations were identified in a proportion exceeding many other series because of the unbiased mutation analysis used, and clinical benefit was seen in some of these. Of 23 different EGFR mutations identified, 11 have not previously been described in the literature. Conclusions: The high prevalence of EGFR, KRAS or both mutations (40%) in this non-squamous population tested in clinical practice supports a policy of routine screening for these mutations in NSCLC.
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Relapse postautograft in acute myeloid leukaemia (AML), may in part arise from leukaemia cells present in the bone marrow (BM) inoculum, and the level of minimal residual disease (MRD) in BM harvests used for autografting may therefore be clinically important. We have used the WT1 transcript as a marker of MRD, which was quantitated by RQ-PCR, in the BM harvests of 24 patients receiving an ABMT for AML. ABL was used as a control gene with WT1 level being normalised to 10(5) copies of ABL per sample. Median WT1 level was 651 copies (range=113-32 700) for the 13 patients with relapse-free survival (RFS) of less than 5 years, and 174 (range=0-1900) for patients with RFS of over 5 years postautograft (P<0.04). The RFS was 10.5 months for patients with WT1 level of >2000 copies (n=5), and has not yet been reached for patients with WT1 level<2000 (n=21), at a median follow-up of 92 months (P<0.05). We show that elevated levels of MRD in BM harvests are associated with a higher relapse risk in patients autografted for AML.
Assuntos
Remoção de Componentes Sanguíneos , Transplante de Medula Óssea , Leucemia Mieloide/terapia , Neoplasia Residual/diagnóstico , RNA Mensageiro/análise , Proteínas WT1/genética , Doença Aguda , Adolescente , Adulto , Medula Óssea/química , Medula Óssea/patologia , Intervalo Livre de Doença , Feminino , Seguimentos , Humanos , Leucemia Mieloide/diagnóstico , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Recidiva , Risco , Transplante AutólogoRESUMO
RT-PCR methods have been developed, to date, by various groups to amplify the PML-RARA fusion gene produced by the t(15;17) in APL patients. However, these methods lack the necessary sensitivity to detect minimal residual disease (MRD) below the level of 1 leukaemic cell in 10(4) cells. Patients who test positive by these methods after treatment are likely to relapse. However, up to 25% of patients who test negative after treatment relapse within a short period. We have developed a 'hot-start' RT-PCR method for the amplification of PML-RARA with increased sensitivity at the level of two leukaemic cells in 10(6) cells. Using this method we were able to detect MRD in seven out of 15 patients tested in remission. Of the 11 patients in medium to long-term remission, five patients tested positive. None of these 11 patients tested positive with the standard RT-PCR. These results show that some patients in remission of APL continue to express PML-RARA even in long-term remission, when they can be considered clinically 'cured' of their disease.
Assuntos
Leucemia Promielocítica Aguda/genética , Proteínas de Neoplasias/análise , Proteínas de Fusão Oncogênica/análise , Reação em Cadeia da Polimerase/métodos , Humanos , Proteínas de Neoplasias/genética , Neoplasia Residual , Proteínas de Fusão Oncogênica/genética , Sensibilidade e Especificidade , Fatores de TempoRESUMO
Qualitative RT-PCR methods used for monitoring minimal residual disease (MRD) in APL patients fail to predict relapse in up to 25% of patients in remission. We report here the development and evaluation of a highly sensitive (10(-5) and 10(-6) with one round and two rounds of PCR, respectively) competitive RT-PCR method to quantitate the PML-RARalpha fusion transcripts. PML-RARalpha transcript's levels were normalised to 10(5) copies of ABL transcript. Serial BM and PB samples from 16 patients with APL and t(15;17) were examined. Presentation samples from three patients (three BM, one PB) showed levels in the range of 0.7 x 10(6)-3.5 x 10(6) and 1.2 x 10(5) molecules in BM and PB samples respectively. Serial quantitation of MRD in both BM and PB samples showed significantly lower levels of PML-RARalpha transcripts in remission, although the majority of samples remain positive for the PML-RARalpha transcripts even those in long-term remission (up to 94 months). Levels of PML-RARalpha in remission samples were up to 2 x 10(2) and up to 5.2 x 10(1) molecules in BM and PB respectively. BM and PB samples taken from two patients 2-4 months before relapse showed significantly higher levels of PML-RARalpha transcripts (1.2 x 10(4) molecules in BM; 3.5 x 102, 1.2 x 10(2) and 1.2 x 10(3) in PB). The same samples, when tested with a standard qualitative RT-PCR for the amplification of PML-RARalpha (with a sensitivity of 10(-4)) produced negative results. This indicates that the qualitative methods would not have predicted relapse in these patients. Our data show that quantitating PML-RARalpha transcripts with a sensitive method may provide a superior approach for monitoring MRD in APL and identifying patients at high risk of relapse.
Assuntos
Leucemia Promielocítica Aguda/diagnóstico , Proteínas de Neoplasias/genética , Proteínas de Fusão Oncogênica/genética , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Humanos , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/metabolismo , Neoplasia Residual , Recidiva , Reprodutibilidade dos Testes , Translocação GenéticaRESUMO
The configuration of immunoglobulin (Ig) and T-cell receptor (TCR) genes was investigated in a case of atypical chronic myeloid leukemia (aCML) lacking a cytogenetically detectable Philadelphia chromosome and molecular evidence of breakpoint cluster region (bcr) rearrangement as determined by 5' and 3' bcr gene probes. Dual clonal rearrangement of Ig-heavy (H) and TCR-delta chain genes was identified. The genes for TCR-gamma and beta chain as well as Ig-kappa (k) chain were in germline configuration. These findings indicate transformation to have occurred in a hemopoietic stem cell with the capacity for antigen receptor gene rearrangement. The demonstration of cross-lineage rearrangement of both Ig and TCR genes lends support to evidence from G6PD alloenzyme studies that the target of transformation in aCML is an early stem cell not yet irreversibly committed to myeloid differentiation. Further studies are indicated to ascertain whether antigen receptor gene rearrangement constitutes a molecular marker useful in the diagnosis of aCML.
Assuntos
Proteínas de Bactérias , Rearranjo Gênico/genética , Imunoglobulinas/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Receptores de Antígenos/genética , Idoso , Alelos , Sondas de DNA , DNA de Neoplasias/metabolismo , Desoxirribonuclease BamHI/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Masculino , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T gama-deltaRESUMO
The (8;21) chromosomal translocation occurs in 20% of adult patients with AML M2. This translocation interrupts two genes, AML1 on chromosome 21q and MTG8 (ETO) on 8q to form a chimeric gene AML1/MTG8 on the der(8) chromosome. Recent reports have shown the presence of diverse forms of transcript for this chimeric gene. Three alternative out-of-frame transcripts have been previously demonstrated (types II, III, IV) all of which have a stop codon 3' of the runt box encoding a truncated runt polypeptide. We have characterized a novel transcript (V) which is in-frame and has a stop codon 3' to the runt box. We have examined transcript diversity in 10 AML patients with t(8;21) in remission of their disease following chemotherapy or bone marrow transplantation. Specific transcripts detected at presentation in six patients were similarly expressed during remission and at relapse in two patients; thus expression of transcript diversity was unaffected by the disease phase. Alternative transcripts were unhelpful as a marker of remission quality or predictor of relapse. The significance of these diverse transcripts in leukemogenesis remains unknown.
Assuntos
Cromossomos Humanos Par 21 , Cromossomos Humanos Par 8 , Proteínas de Ligação a DNA/genética , Leucemia Mieloide Aguda/genética , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogênicas , Fatores de Transcrição/genética , Transcrição Gênica , Translocação Genética , Sequência de Bases , Subunidade alfa 2 de Fator de Ligação ao Core , Expressão Gênica , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Dados de Sequência Molecular , Proteína 1 Parceira de Translocação de RUNX1 , Recidiva , Indução de RemissãoRESUMO
We studied 41 patients with myelodysplastic syndromes or acute myeloid leukemia to assess the presence of point mutations in the human FMS gene (M-CSF receptor). Using the polymerase chain reaction and hybridization of oligonucleotide probes to the amplified sequences, we have detected mutations in eight of 41 patients, at codons 301 and 969. In vitro work has highlighted mutations at these codons as being oncogenic. We now report the detection of potentially activating mutations of the human FMS gene in vivo. The consequence of these mutations in the multistep pathogenesis of myeloid malignancy and their relevance to prognosis remains to be determined.
Assuntos
Leucemia Mieloide Aguda/genética , Síndromes Mielodisplásicas/genética , Proteínas Proto-Oncogênicas/genética , Alelos , Sequência de Bases , Códon/genética , Humanos , Dados de Sequência Molecular , Mutação , Hibridização de Ácido Nucleico , Oligonucleotídeos/genética , Reação em Cadeia da Polimerase , Receptor de Fator Estimulador de Colônias de MacrófagosRESUMO
We have developed a competitor-based RT-PCR technique which will detect and quantitate the CBFbeta/MYH11 transcripts associated with inv(16)(q22;p13) and have used it to study presentation and follow-up samples of acute myeloid leukaemia (AML). The levels of the leukaemia-specific transcripts are expressed as a ratio to a ubiquitously expressed mRNA species (Abl) which controls for RNA degradation. This technique has been applied to 75 consecutive patients presenting with either de novo AML or tMDS; 6/75 patients analysed were positive for the inv(16), all were confirmed by conventional cytogenetics. The inv(16) has a strong association with M4Eo, but we found only 2/6-positive patients to have this diagnosis (two patients with M2, one patient M1 and one patient had MDS). At presentation the levels of CBFbeta/MYH11 transcripts were 0.1-10/Abl transcript (mean 3.3/Abl transcript). Seventeen follow-up samples were available on 5/6 of these patients, and on two further patients in whom stored material was available. Following the first cycle of chemotherapy the level of transcripts was at least 10(-2) lower (0.1-10 x 10(-2)/abl transcript) than their presentation sample. Subsequent samples on these patients when in remission gave transcript levels in the range (1.0 x 10(-4) - 2 x 10(-3)/abl transcript), and three long-term follow-up samples were negative. We have developed a quantitative test which opens the possibility of predicting relapse by detecting changes in the numbers of leukaemia-specific transcripts.
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Inversão Cromossômica , Cromossomos Humanos Par 16 , Leucemia Mieloide/genética , Leucemia Mieloide/metabolismo , Proteínas de Fusão Oncogênica/biossíntese , Proteínas de Fusão Oncogênica/genética , Doença Aguda , Adulto , Idoso , Criança , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase/métodos , Transcrição GênicaRESUMO
PURPOSE: Uveal melanoma is the most common intraocular malignancy in adults and can cause loss of vision in the affected eye and death from metastasis, usually to the liver. The techniques currently used to detect cellular dissemination from the tumor are inadequate, and lack the sensitivity required for the detection of low levels of melanocytes in the peripheral blood of patients. The detection of circulating melanocytes is important as an early indication of the possibility of metastasis. METHODS: The viability of reverse transcription/polymerase chain reaction amplification of the tyrosinase gene to detect circulating melanocytes was examined as a first sign of dissemination from uveal melanoma. RESULTS: It was shown that it is possible to detect as few as ten circulating melanocytes in 5 ml of blood. Blood-borne dissemination was also detected in three of six patients with uveal melanoma examined. Two of these patients had clinically confirmed widespread metastases. A positive result was also recorded in one patient in whom there was no other evidence for tumor dissemination. Overt metastatic disease developed in this patient 9 months after blood collection. CONCLUSIONS: The success of this technique has important implications for the detection of circulating tumor cells from uveal melanoma, as an early indication of dissemination. This may be important when considering the administration of adjuvant therapy.
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Melanócitos , Melanoma/diagnóstico , Melanoma/secundário , Células Neoplásicas Circulantes , Reação em Cadeia da Polimerase/métodos , Neoplasias Uveais/sangue , Adulto , Idoso , Sequência de Bases , Pré-Escolar , Eletroforese em Gel de Poliacrilamida , Feminino , Humanos , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/secundário , Melanócitos/enzimologia , Melanoma/sangue , Pessoa de Meia-Idade , Dados de Sequência Molecular , Monofenol Mono-Oxigenase/genética , Monofenol Mono-Oxigenase/metabolismo , RNA Mensageiro/análise , Células Tumorais CultivadasRESUMO
Seven patients with acute myeloid leukaemia (AML) type M2 and t(8;21), treated with intensive chemotherapy followed in two cases by allogeneic or autologous bone marrow transplant (BMT), were tested for the presence of transcripts of the characteristic chimaeric gene AML1/ETO by reverse transcriptase polymerase chain reaction (RT-PCR) at serial intervals during remission. Six patients, including both BMT patients, demonstrated persistence of t(8;21), one being consistently negative in peripheral blood but bone marrow positive. Bone marrow may, therefore, be more reliable than peripheral blood for detecting residual t(8;21) cells. Our results show persistence of t(8;21) cells in AML-M2 patients following chemotherapy or BMT, some of whom were in long-term remission.
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Leucemia Mieloide Aguda/diagnóstico , Adulto , Idoso , Sequência de Bases , Transplante de Medula Óssea , Aberrações Cromossômicas/diagnóstico , Transtornos Cromossômicos , Cromossomos Humanos Par 21 , Cromossomos Humanos Par 8 , Primers do DNA , Feminino , Humanos , Leucemia Mieloide Aguda/terapia , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/métodos , RNA Mensageiro/análise , RNA Neoplásico/análise , Translocação GenéticaRESUMO
Delayed post-operative endophthalmitis is a complication of modern cataract extraction and posterior chamber lens implantation. Propionibacterium acnes has been isolated in a few such cases but the majority are culture-negative, compounding surgical and medical management decisions. A method of detecting bacterial, and specifically P. acnes, DNA by the polymerase chain reaction (PCR) directed at 16S rDNA is reported. Nested PCR with universal eubacterial primers complimentary to regions of 16S rDNA conserved sequences detected 50 fg of bacterial DNA spike in normal vitreous. Nested PCR with P. acnes primers detected 10 fg of DNA. Vitreous samples from 29 patients undergoing vitrectomy for reasons unrelated to infection and 23 samples from 19 patients with delayed post-operative endophthalmitis were analysed. Four (14%) of 29 normal individuals and 17 (74%) of 23 delayed cases gave positive results with universal eubacterial primers. None of 29 and eight of 23 samples gave positive results with P. acnes primers. The 14% positive rate with universal primers in non-infected cases may limit their use in delayed post-operative endophthalmitis. PCR detection of bacterial DNA with specific primers from vitreous samples may prove a useful means of diagnosing delayed post-operative endophthalmitis and facilitating management decisions when conventional bacterial culture is negative.
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Endoftalmite/diagnóstico , Infecções Oculares Bacterianas/diagnóstico , Infecções por Bactérias Gram-Positivas/diagnóstico , Complicações Pós-Operatórias/diagnóstico , Propionibacterium acnes/isolamento & purificação , Sequência de Bases , Sequência Conservada , Primers do DNA/química , DNA Bacteriano/análise , DNA Bacteriano/química , DNA Ribossômico/análise , DNA Ribossômico/química , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Propionibacterium acnes/genética , RNA Ribossômico 16S/genética , Sensibilidade e Especificidade , Vitrectomia , Corpo Vítreo/microbiologiaRESUMO
The t(8;21) is one of the most common translocations in acute myeloid leukaemia (AML) occurring in approximately 20% of adult and 40% of paediatric AML-M2. This translocation fuses the AML1 gene on chromosome 21q to the MTG8 (ETO) gene on chromosome 8q to produce the fusion gene AML1-MTG8. Transcripts for the AML1-MTG8 fusion gene have been detected in the majority of patients in remission by qualitative RT-PCR methods. Thus for such patients these methods are unsuitable for monitoring minimal residual disease (MRD). Furthermore, the diverse form of transcripts for this fusion gene was found in patients at different phases of their disease, which rules out the usefulness of the expression of any particular set of transcripts as a marker for monitoring MRD in those patients. On the other hand a quantitative RT-PCR method we developed, was able to assess the effectiveness of treatment and predict relapse up to four months before the onset of haematological relapse. This method should distinguish patients in stable remission from those at high risk of relapse and therefore identify patients who would require additional or new treatment such as BMT.
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Cromossomos Humanos Par 21 , Cromossomos Humanos Par 8 , Leucemia Mieloide Aguda/genética , Monitorização Fisiológica/métodos , Neoplasia Residual/genética , Translocação Genética , Adulto , Criança , Humanos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e EspecificidadeRESUMO
An immunohistochemical double staining technique was used to examine the characteristics of cellular infiltration in choroidal melanoma. Seven of 16 melanomas examined demonstrated high levels of cellular infiltration, mainly with T-cells and macrophages, and little infiltration with B-cells and NK cells. The majority of T-cells were of the CD8+ type and were activated, as shown by the expression of histocompatibility antigens, HLA-DR and IL2-R. Most of the infiltrating macrophages also expressed HLA-DR antigen. We also detected malignant melanocytes expressing the HLA-DR antigen. This technique could be used to study in detail cellular infiltration in a large number of archival choroidal melanomas with known clinical history, which would enable detection of markers that correlate with the prognosis of the disease.
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Subpopulações de Linfócitos B/patologia , Neoplasias da Coroide/patologia , Linfócitos do Interstício Tumoral/patologia , Melanoma/patologia , Subpopulações de Linfócitos T/patologia , Complexo CD3/análise , Antígenos CD4/análise , Antígenos CD8/análise , Neoplasias da Coroide/imunologia , Antígenos HLA-DR/análise , Humanos , Células Matadoras Naturais/patologia , Melanoma/imunologia , Receptores de Interleucina-2/análiseRESUMO
Reliable detection of JAK2-V617F is critical for accurate diagnosis of myeloproliferative neoplasms (MPNs); in addition, sensitive mutation-specific assays can be applied to monitor disease response. However, there has been no consistent approach to JAK2-V617F detection, with assays varying markedly in performance, affecting clinical utility. Therefore, we established a network of 12 laboratories from seven countries to systematically evaluate nine different DNA-based quantitative PCR (qPCR) assays, including those in widespread clinical use. Seven quality control rounds involving over 21,500 qPCR reactions were undertaken using centrally distributed cell line dilutions and plasmid controls. The two best-performing assays were tested on normal blood samples (n=100) to evaluate assay specificity, followed by analysis of serial samples from 28 patients transplanted for JAK2-V617F-positive disease. The most sensitive assay, which performed consistently across a range of qPCR platforms, predicted outcome following transplant, with the mutant allele detected a median of 22 weeks (range 6-85 weeks) before relapse. Four of seven patients achieved molecular remission following donor lymphocyte infusion, indicative of a graft vs MPN effect. This study has established a robust, reliable assay for sensitive JAK2-V617F detection, suitable for assessing response in clinical trials, predicting outcome and guiding management of patients undergoing allogeneic transplant.
Assuntos
Janus Quinase 2/genética , Mutação/genética , Transtornos Mieloproliferativos/genética , Recidiva Local de Neoplasia/diagnóstico , Neoplasia Residual/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real , Adulto , Idoso , Análise Citogenética , Europa (Continente) , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Transtornos Mieloproliferativos/terapia , Recidiva Local de Neoplasia/genética , Neoplasia Residual/genética , Prognóstico , RNA Mensageiro/genética , Indução de Remissão , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transplante de Células-Tronco , Transplante Homólogo , Adulto JovemRESUMO
Quantitative PCR (qPCR) for detection of fusion transcripts and overexpressed genes is a promising tool for following minimal residual disease (MRD) in patients with hematological malignancies. Its widespread clinical use has to some extent been hampered by differences in data analysis and presentation that complicate multicenter clinical trials. To address these issues, we designed a highly flexible MRD-reporting software program, in which data from various qPCR platforms can be imported, processed, and presented in a uniform manner to generate intuitively understandable reports. The software was tested in a two-step quality control (QC) study; the first step involved eight centers, whose previous experience with the software ranged from none to extensive. The participants received cDNA from consecutive samples from a BCR-ABL+ chronic myeloid leukemia (CML) patient and an acute myeloid leukemia (AML) patient with both CBFß-MYH11 and WT1 target genes, they conducted qPCR on their respective hardware platforms and generated a series of reports with pre-defined features. In step two, five centers used the software to report BCR-ABL+ MRD in a harmonized manner, applying their recently obtained CML international scale conversion factors. The QC study demonstrated that this MRD-reporting software is suitable for efficient handling of qPCR data, generation of MRD reports and harmonization of MRD data.
Assuntos
Sistemas de Gerenciamento de Base de Dados , Bases de Dados Genéticas , Neoplasia Residual/genética , Relatório de Pesquisa/normas , Reação em Cadeia da Polimerase Via Transcriptase Reversa/estatística & dados numéricos , DNA Complementar/genética , DNA de Neoplasias/genética , Europa (Continente)/epidemiologia , Genes do Tumor de Wilms , Humanos , Serviços de Informação , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mieloide Aguda/genética , Neoplasia Residual/epidemiologia , Proteínas de Fusão Oncogênica/genética , Controle de Qualidade , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Pesquisa Translacional Biomédica/métodosRESUMO
We have developed a quantitative reverse transcriptase-polymerase chain reaction method for the quantitation of AML1-MTG8 transcripts in patients with AML-M2 and t(8;21) in different phases of the disease. Using this method, we have tested sequential samples from 13 patients to monitor minimal residual disease and were able to show a significant increase in AML1-MTG8 transcripts level in two patients 2 and 4 months before clinical relapse. In five patients tested at presentation and then sequentially at remission, we detected a marked decrease in the level of AML1-MTG8 transcripts as the treatment progressed. Patients in long-term remission of their disease had a level of up to 1 x 10(3) AML1-MTG8 molecules/microgram RNA. Two patients tested 2 and 4 months before hematologic relapse showed a level of 0.71 x 10(5) molecules/microgram RNA and this level increased further during relapse to 0.71 x 10(7) and 2.27 x 10(5) molecules/microgram RNA, respectively. Our results show that quantitation of AML1-MTG8 transcripts by competitive polymerase chain reaction is valuable in predicting early relapse in AML with t(8;21). Identification of at-risk patients may allow treatment to be modified to include additional or alternative therapy such as bone marrow transplantation.
Assuntos
Cromossomos Humanos Par 21/genética , Cromossomos Humanos Par 8/genética , Proteínas de Ligação a DNA/genética , Leucemia Mieloide Aguda/patologia , Proteínas de Fusão Oncogênica/genética , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas , RNA Mensageiro/análise , RNA Neoplásico/análise , Fatores de Transcrição/genética , Translocação Genética , Adolescente , Adulto , Exame de Medula Óssea , Cromossomos Humanos Par 21/ultraestrutura , Cromossomos Humanos Par 8/ultraestrutura , Subunidade alfa 2 de Fator de Ligação ao Core , Proteínas de Ligação a DNA/biossíntese , Intervalo Livre de Doença , Feminino , Regulação Leucêmica da Expressão Gênica , Humanos , Leucemia Mieloide Aguda/genética , Masculino , Pessoa de Meia-Idade , Neoplasia Residual , Prognóstico , Proteína 1 Parceira de Translocação de RUNX1 , Risco , Sensibilidade e Especificidade , Fatores de Transcrição/biossínteseRESUMO
Using CM-1 antibody directed against the human p53 protein, high levels of mutant p53 protein expression were found in 12 out of 18 malignant choroidal melanomas. In contrast, we failed to observe elevated p53 expression, indicating the absence of p53 mutation in seven choroidal naevi, a potentially premalignant condition that can progress to form malignant melanoma. For two choroidal melanomas, we demonstrated that high levels of p53 protein were accompanied by exon 7 mutations. The mutations were found at codon 238, TGT-->TTT and codon 253, ACC-->AGC. These observations suggest that acquisition of abnormalities of the p53 gene may be an important step in the development of malignant melanoma.
Assuntos
Neoplasias da Coroide/genética , Expressão Gênica/genética , Genes p53/genética , Melanoma/genética , Animais , Anticorpos , Sequência de Bases , Western Blotting , Humanos , Dados de Sequência Molecular , Mutação , Nevo/genética , Nevo/metabolismo , Coelhos , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/imunologia , Proteína Supressora de Tumor p53/metabolismoRESUMO
Acute promyelocytic leukaemia (APL) is characterized by t(15;17), which results in the formation of two chimaeric genes, PML-RAR alpha and RAR alpha-PML. PML-RAR alpha transcripts have been detected in all cases of APL whilst those of RAR alpha-PML have been detected in only about 67% of cases. We have used reverse transcriptase polymerase chain reaction (RT-PCR) to detect both fusion transcripts serially in 18 patients in remission of APL after chemotherapy and bone marrow transplantation. All patients were negative for PML-RAR alpha, whereas in six patients (remission 3-9 years) RAR alpha-PML was consistently detected. Only one patient at remission showed the 5' breakpoint RAR alpha-PML, with the rest showing the 3' breakpoint 144 bp RAR alpha-PML. The level of sensitivity for detecting RAR alpha-PML was some 10-fold higher than that for PML-RAR alpha. Serial negative tests for PML-RAR alpha have been correlated with durable remissions, suggesting possible eradication of residual leukaemia in APL. Our results, however, show persistence of t(15;17) cells expressing RAR alpha-PML fusion mRNA in patients in long-term remission of APL. They indicate that patients considered clinically 'cured' of APL still have molecular evidence of minimal residual disease and also provide further insight into the biology of acute myeloid leukaemia.